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1.
Figure 9

Figure 9. From: Modulation of synaptic function by VAC14, a protein that regulates the phosphoinositides PI(3,5)P2 and PI(5)P.

Model of trafficking defects that promote the elevation of surface AMPA receptors in Vac14−/− neurons. Endocytosis of surface GluA2 is diminished in Vac14−/− neurons compared with wild type (1), while no defects were detected in recycling of internal GluA2 to the cell surface (2) or membrane transport late in the endocytic pathway (3). Note that in the absence of VAC14, FIG4 is destabilized (Lenk et al, 2011). EE, early endosomes; LE, late endosomes; RE, recycling endosomes.

Yanling Zhang, et al. EMBO J. 2012 August 15;31(16):3442-3456.
2.
Figure 5

Figure 5. From: Modulation of synaptic function by VAC14, a protein that regulates the phosphoinositides PI(3,5)P2 and PI(5)P.

Restoration of VAC14 eliminates the increase in mEPSC amplitude in Vac14−/− neurons. (A) Examples of mEPSC recordings of wild-type and Vac14−/− neurons that were sham-transfected, expressed VAC14-Citrine or Citrine alone. (B) Quantification of mEPSC amplitude normalized to sham-transfected wild-type neurons shows that there is an inverse relationship between level of VAC14 expression and mEPSC amplitude. In wild-type neurons, VAC14 overexpression leads to a decrease in amplitude. The increase in mEPSC amplitude in Vac14−/− neurons is reduced by re-introduction of VAC14. Neurons transfected with Citrine were similar to untransfected wild type. One-way ANOVA test was used to compare mEPSC amplitudes (F(5, 12.3), P=8.27 × 10−9). *Individual comparisons between untransfected and VAC14-transfected wild-type and untransfected and VAC14-transfected Vac14−/− neurons were significant using Tukey-Kramer honestly significant difference (HSD) criterion. WT, wild type; KO, Vac14−/−. Error bars, s.e.m.

Yanling Zhang, et al. EMBO J. 2012 August 15;31(16):3442-3456.
3.
Figure 2

Figure 2. From: Modulation of synaptic function by VAC14, a protein that regulates the phosphoinositides PI(3,5)P2 and PI(5)P.

VAC14 is found in both dendrites and axons, and colocalizes with endocytic and synaptic markers in hippocampal neurons. (A) Wild-type and Vac14−/− neurons were double labelled with rabbit anti-VAC14 and mouse anti-MAP2 (dendrites) or mouse anti-TAU-1 (axons). Arrows indicate the localization of VAC14 on dendrites (MAP2-positive and TAU-negative neurites). Arrowheads indicate the localization of VAC14 on axons (MAP2-negative and TAU-1-positive neurites). Bar=10 μm. (B) VAC14 partially colocalizes with EEA1 or LAMP1 in the neurites (arrows). Wild-type and Vac14−/− neurons were triple labelled with rabbit anti-VAC14, chicken anti-EEA1 and rat anti-LAMP1. Bar=5 μm. (C) VAC14 displays significant colocalization with several synaptic markers: synapsin, synaptotagmin and synaptobrevin. Wild-type and Vac14−/− neurons were double labelled with rabbit anti-VAC14 and guinea pig anti-synapsin, mouse anti-synaptotagmin or mouse anti-synaptobrevin. Bar=5 μm. (D) VAC14 partially localizes at excitatory synapses (labelled with both the synaptic vesicle glutamate transporter vGlut1 and postsynaptic marker PSD95). Wild-type and Vac14−/− neurons were labelled with rabbit anti-VAC14, mouse anti-PSD95 and guinea pig anti-vGlut1. Arrows indicate examples of colocalization. (C, D) Lower panels show straightened dendrites from corresponding top panels. Bar=5 μm.

Yanling Zhang, et al. EMBO J. 2012 August 15;31(16):3442-3456.
4.
Figure 3

Figure 3. From: Modulation of synaptic function by VAC14, a protein that regulates the phosphoinositides PI(3,5)P2 and PI(5)P.

Loss of VAC14 or FIG4 leads to an increase in excitatory synaptic function. (A) Representative mEPSC recordings of wild-type (N=32) and Vac14−/− neurons (N=32). (B) Mean mEPSC amplitude in Vac14−/− neurons is larger than in wild-type neurons, 20.86±1.03 pA versus 16.83±0.91 pA, respectively. *P=0.0045, t-test. (C) Mean mEPSC frequency is similar in wild-type (1.21±0.26 Hz) and Vac14−/− (1.16±0.24 Hz) neurons. (D, E) Summary of mEPSC kinetics in wild-type and Vac14−/− neurons. (D) Individual mEPSCs overlaid. Thick lines show the mean trace. Dashed line is aligned to the mean peak inward current of Vac14−/− mEPSC. Scaled overlay shows similar kinetics between wild-type and Vac14−/− mEPSCs. (E) Mean mEPSC decay is similar between wild type (3.80±0.15 ms) and Vac14−/− (3.69±0.14 ms). (F) Representative mEPSC traces of wild-type (Fig4+/+) (N=12) and Fig4−/− neurons (N=14). (G) Mean mEPSC amplitude in Fig4−/− neurons is larger than in Fig4+/+ neurons (14.64±0.39 pA versus 18.73±1.38 pA, respectively, *P=0.0164, t-test). (H) Mean mEPSC frequency is similar in Fig4+/+ and Fig4−/− (0.99±0.22 Hz versus 0.92±0.28 Hz, respectively, P=0.8542, t-test). Error bars are standard error of the mean (s.e.m.).

Yanling Zhang, et al. EMBO J. 2012 August 15;31(16):3442-3456.
5.
Figure 7

Figure 7. From: Modulation of synaptic function by VAC14, a protein that regulates the phosphoinositides PI(3,5)P2 and PI(5)P.

GluA2 endocytosis is reduced in Vac14−/− hippocampal neurons. (A) Diagram of experiment. Wild-type or Vac14−/− hippocampal neurons were treated with the lysosomal protease inhibitor leupeptin, then live labelled with mouse GluA2 antibodies to label those receptors exposed to the cell surface. Endocytosis was stimulated with 50 μM NMDA for 10 min. Then any remaining surface-exposed GluA2 antibody was removed with an acid wash. Neurons were then fixed and labelled with Alexa 555 anti-mouse IgG. (B) Example of internalized GluA2. Intensity presented in the ‘fire' LUT colour scheme. Bar=10 μm. (C) Total internalized GluA2 in the soma was decreased in Vac14−/− neurons (N=55 for wild type and 42 for Vac14−/−, *P=2.73 × 10−5, t-test). (D) Total internalized GluA2 in the dendrites was decreased in Vac14−/− neurons (N=57 for wild type and 61 for Vac14−/−, *P=8.33 × 10−5, t-test). (E) The number of internalized GluA2 puncta were decreased in Vac14−/− neurons (N=57 for wild type and 61 for Vac14−/−, *P=5.91 × 10−8, t-test). A fixed length (35 μm from the soma) was used for dendrites in (D) and (E). Error bars, s.e.m.

Yanling Zhang, et al. EMBO J. 2012 August 15;31(16):3442-3456.
6.
Figure 1

Figure 1. From: Modulation of synaptic function by VAC14, a protein that regulates the phosphoinositides PI(3,5)P2 and PI(5)P.

Endogenous VAC14 partially colocalizes with multiple endocytic organelles. (A) Polyclonal VAC14 antibody recognizes punctate structures in wild-type cells. Fibroblasts were permeabilized with saponin followed by fixation, then labelled with anti-VAC14 antibody. Bottom panels, DIC images. (B) In fibroblasts, endogenous VAC14 colocalizes with both EEA1 and LAMP1. Wild-type fibroblasts were triple labelled with rabbit anti-VAC14, chicken anti-EEA1 and rat anti-LAMP1. The majority of VAC14 colocalized with either EEA1 (yellow arrows) or LAMP1 (turquoise arrows). Some VAC14 colocalized with both (white arrows) or neither (green arrow) markers. (C) VAC14 partially colocalizes with the late endosome marker LBPA (arrow). Fibroblasts were double labelled with rabbit anti-VAC14 and mouse anti-LBPA. (D) VAC14 partially colocalized with lysosomes (arrow). To label lysosomes, prior to fixation, fibroblasts were pulsed with Texas Red-Dextran (Mw 70 kD) for 1 h and chased in the absence of dextran for 24 h. (E) The limiting membrane of vacuoles in Vac14−/− cells is positive for LAMP1 while negative for LBPA, suggesting a lysosomal origin. Vac14−/− fibroblasts were double labelled with rat anti-LAMP1 and mouse anti-LBPA. (D, E) DAPI (blue) used to label nuclei. (F) VAC14 partially colocalizes with LC3-RFP puncta (arrows). Fibroblasts transfected with LC3-RFP were fixed and labelled with anti-VAC14. (AF) Bar=10 μm.

Yanling Zhang, et al. EMBO J. 2012 August 15;31(16):3442-3456.
7.
Figure 6

Figure 6. From: Modulation of synaptic function by VAC14, a protein that regulates the phosphoinositides PI(3,5)P2 and PI(5)P.

Surface GluA2 levels increase in Vac14−/− neurons. (A) Surface GluA2 was labelled by incubation of intact neurons with mouse anti-GluA2 antibody. Arrows highlight the dendrite used for analysis (enlarged in lower panels). Intensity presented in the ‘fire' LUT colour scheme. (B) Quantitation of the intensity of GluA2 puncta, normalized to the wild-type mean. The relative intensity values from wild-type and Vac14−/− neurons are presented as a cumulative distribution. A Kolmogorov-Smirnov test demonstrates that the data sets differ significantly; P=1.1 × 10−34. The median of each data set also differ significantly (box plot shown in insert). Box, interquartile range; line, median; square, mean; non-overlapping notches indicate that the two medians are statistically different at the 5% significance level; whiskers, minimum and maximum of the data within 1.5 times the length of the box. N=5272 for wild type and 4756 for Vac14−/−. Error bars, s.e.m. (C) Surface GluA2 subunits accumulate on the surface of Vac14−/− dendrites. Top three panels (wild type) and middle three panels (Vac14−/−) dendrites (left to right) show surface GluA2, total GluA2, and the dendritic marker, MAP2. Yellow arrows highlight dendrites analysed. Bottom panels show the merged image of the straightened dendrite (left) and MAP2 (right). (D) The ratio of surface to total GluA2 is increased in Vac14−/− dendrites (1.32±0.052) relative to wild type (1.0±0.0364). *P=6.836 × 10−7, two-sample t-test. Scale bar=10 μm. Error bars=s.e.m.

Yanling Zhang, et al. EMBO J. 2012 August 15;31(16):3442-3456.
8.
Figure 8

Figure 8. From: Modulation of synaptic function by VAC14, a protein that regulates the phosphoinositides PI(3,5)P2 and PI(5)P.

Endocytosis of AMPA receptors is reduced in Vac14−/− neurons. Cultured hippocampal neurons were transfected with pH-GluA1and mCherry at DIV12. At DIV14, cells were placed on the microscope stage and perfused with normal extracellular buffer. Images were acquired once per minute for a 10-min baseline, then switched to NMDA stimulation buffer for 5 min to stimulate internalization. Then, following replacement of the NMDA stimulation buffer with normal buffer, monitored for recycling back to the cell surface. The pHluorin fluorescence was imaged at 488 nm excitation, while mCherry fluorescence was imaged at 559 nm excitation, through a × 60 oil objective at a rate of one image per minute. (A) Representative full-frame images of wild-type and Vac14−/− neurons during baseline (0–10 min), NMDA stimulation (11–15 min) and recovery after wash out (16–55 min). (B) Changes in pH-GluA1 fluorescence were calculated from straightened dendrites isolated from full-frame images. (C) Wild-type and Vac14−/− neurons exhibited similar recovery to baseline levels. (D) Average time course for percent change in pH-GluA1 fluorescence, normalized to average baseline intensity. (E) Amplitude of change in fluorescence after wash-out of NMDA is decreased in Vac14−/− neurons compared to wild type (wild type, 66.72±4.93%; Vac14−/− 44.68±7.10%). (F) t1/2 recycling rate after NMDA removal is normal. *P=0.0224, Two-sample t-test, n=11–14. Error bars, s.e.m.

Yanling Zhang, et al. EMBO J. 2012 August 15;31(16):3442-3456.
9.
Figure 4

Figure 4. From: Modulation of synaptic function by VAC14, a protein that regulates the phosphoinositides PI(3,5)P2 and PI(5)P.

Presynaptic probability of release is enhanced in Vac14−/− neurons. (A, B) Synaptic vesicles from Vac14−/− are not larger than synaptic vesicles observed in brains from wild type. (A) Electron microscopy of excitatory synapses, evident by the thickening of the postsynaptic membrane, in wild-type and Vac14−/− hippocampus and hindbrain. Bar=100 nm. (B) Quantitation of the diameter of synaptic vesicles. Cumulative probability distribution of synaptic vesicle diameter. No significance difference was found between wild type and Vac14−/− by a two-sample Kolmogorov-Smirnov test (kstest2, Matlab) (hippocampus, P=0.32; hindbrain, P=0.46). Three wild-type and three Vac14−/− animals were analysed. Hindbrain: N=567 vesicles from 33 terminals for wild type and 388 vesicles from 29 terminals for Vac14−/−. Hippocampus: N=433 vesicles from 33 terminals for wild type and 66 vesicles from 15 terminals for Vac14−/−. (C, D) The number of synapses is decreased in Vac14−/− neurons. (C) Wild-type and Vac14−/− hippocampal neurons were triple labelled with rabbit anti-MAP2 (blue), mouse anti-PSD95 (red) and guinea pig anti-vGlut (green). Examples of straightened dendrites are shown. Bar=5 μm. (D) Quantitation of the number of synapses on the first 100 μm of dendrites starting from the soma. The numbers of synapses were normalized to the average of wild type. Vac14−/− neurons had fewer synapses (N=91 for wild type and 71 for Vac14−/−) (*P=1.7 × 10−4, t-test). Error bars, s.e.m. (E, F) Presynaptic probability of release is increased in Vac14−/− neurons. NMDA currents were pharmacologically isolated from AMPA and GABAA mediated currents and recorded at −70 mV (solution contained zero Mg2+). An extracellular stimulating electrode was placed locally and used to stimulate vesicle release in afferent axons. Once a stable response was obtained, 20 μM MK801, an open-channel blocker of NMDA receptors, was added to the bath for 5 min without stimulation. Then, 200 stimulations were delivered and amplitude of the current measured (mean amplitude is shown by filled squares). In the presence of MK-801, the current is progressively blocked. The data for each genotype were fitted with a double exponential curve. The rate of progressive blockade of NMDA current was significantly greater in Vac14−/− neurons; mean amplitude of the 2–11 stimulations is significantly lower in Vac14−/− neurons. *P=0.0217, Anova1 (Matlab).

Yanling Zhang, et al. EMBO J. 2012 August 15;31(16):3442-3456.

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